The long-range objective of this research program is to understand in molecular terms how the centromere-kinetochore functions in eukaryotic cell division. Our previous research in this area emphasized centromere structure/function studies in the budding yeast Saccharomyces cerevisiae and fission yeast Schizo-saccharomyces pombe. Building on the valuable information gained in these groundwork studies, we will now extend the work to investigate centromere-kinetochores in the two most common and clinically significant pathogenic budding yeasts, Candida albicans and Candida glabrata. All budding yeast species examined to date contain relatively small point centromeres (CEN less than 400 bp in length), whereas other organisms contain large regional centromeres characterized by the presence of long stretches of heterochromatic repeated DNA sequences. It is postulated that this fundamental difference in centromere structure could be exploited eventually to develop agents selectively toxic to the budding yeasts. Specific aims are: (1) combined molecular and genetic strategies will be used to isolate and characterize CEN DNAs of C. albicans; (2) stable CEN-based plasmid and artificial chromosome vector systems will be constructed to facilitate molecular genetic research in C. albicans; and (3) inner kinetochore-associated proteins from both C. albicans and C. glabrata will be identified and characterized as potential targets for specific antifungal drug therapy. We have identified, epitope-tagged, and expressed an evolutionarily conserved, centromere-specific histone H3 protein (CaCse4p) in C. albicans. Chromatin immunoprecipitation (CHIP) with antibodies directed against this protein will be used to isolate and clone CEN DNAs. Putative CEN DNAs will be identified and characterized in terms of their nucleotide sequence and ability to mitotically stabilize plasmid and artificial chromosome vectors in actively dividing cells. Inner kinetochore proteins of Candida species will be isolated by CEN DNA affinity chromatography and the corresponding genes will be cloned and characterized. One-hybrid and two-hybrid screens currently under development for use with C. glabrata will be applied to a search for additional genes specifying CEN-associated proteins. Kinetochore association of candidate proteins will be confirmed by ChIP analysis and by studying the effects of gene deletion and/or mutagenesis on cell division and chromosome segregation.